Only very limited examples for the stabilization of frozen liquid viable virus vaccines at −20° C. have been reported. Most of these did not employ purified viable enveloped virus (1, 2, 3, 4, 5). One of the major challenges to stabilizing enveloped virus at temperatures below the freezing point is preventing the physical disruption of structural and functional components (i.e. proteins, lipid bilayer and virus genome) during the freezing and storage stages. It has been reported that proteins are susceptible to denaturation (6), and lipid bilayers are prone to rupture during freezing (7). Several types of excipients have been reported to stabilize the structure of the lipid bilayer and proteins during freezing and in the frozen state (6, 7). These excipients include: polyols, saccharides, buffers, amino acids, and polymers.
The major tasks for stabilization of enveloped virus at temperatures below the freezing point are preventing the physical disruption of the virus's structural and functional components during both the freezing and storage stages. The enveloped virus components include: 1) the lipid bilayer envelope membrane; 2) the proteins coded by the viral genome, and 3) the single-stranded, or double stranded DNA or RNA genome.
In order to ensure stability during storage, stocks of infective virus have commonly been stored at ultra-low temperature (e.g. at ≦−60° C.) due to their complexity. Gould, E. A. (“Methods for long-term Virus Preservation”, Molecular Biotechnology Vol. 13, pp. 57-66, 1999) teach that lipid enveloped viruses survive well at ultra low temperatures below −60° C. but that storage at −20° C. should only be used if “retention of virus infectivity is not essential”. D. R. Harper described the storage conditions for a wide variety of non-enveloped and enveloped viruses (“Virology, Ed. D. R. Harper, BIOS Scientific Publishers Limited, Oxford, UK, 1993). In all cases, viruses must be stored at either −70° C. in liquid form or at 4° C. as a lyophile in order to retain infectivity. The storage conditions for liquid formulations of Newcastle disease virus are specifically mentioned as −70° C.
Yannarell et al (“Stabilizing cold-adapted influenza virus vaccine under various storage conditions”, J. Virol. Meth. Vol. 102, pp. 15-25, 2002) describe conditions for storage of cold-adapted influenza virus vaccine at −20° C. using SPG, a mixture of sucrose, phosphate and glutamate (0.218M sucrose, 0.0038M monobasic potassium phosphate, 0.0072M dibasic potassium phosphate, 0.0049M potassium glutamate). Influenza virus prepared in allantoic fluid for intranasal administration was diluted 10% with a 10× solution of SPG. The final concentration of sucrose in this mixture was 7.5%. The presence of phosphate does not help to stabilize NDV while glutamate hinders sterile filtration and thus both compounds are detrimental to NDV preparation and storage at −20° C.
Parenteral administration adds an additional formulation issue. For safety reasons products for parenteral usage must be sterile filtered through a 0.2 μm filter, as terminal sterilization is not possible for viable virus preparations. Newcastle disease virions are pleomorphic but roughly spherical particles ranging in approximate size from 0.1 to 0.5 μm in diameter. The recovery rate of NDV filtered through a 0.2 μm sterile filter is formulation dependent and an important factor to be considered for developing a −20° C. liquid NDV formulation.
The factors affecting the ability of NDV to pass through a 0.2 μm sterile filter include the diameter of the virus, the filter pore size and the adsorptivity of NDV to the filter. The apparent diameter of NDV can be affected by: 1) The tonicity of the formulation; and 2) the surface charge of NDV, which may affect the molecular configuration and adsorption of proteins or nucleic acid on the surface of NDV in the presence of different buffers.
Adsorption of NDV to the filter membrane may also have a significant effect on the ability of the virus to sterile filter. Several factors may have impact on the surface properties of NDV and thus affect the adsorptivity of NDV to the filter. These factors include: 1) pH, 2) ionic strength, 3) surface interactions including hydrophobic or Van Der Waal interactions and ionic interactions and 4) the presence of surface-active agents such as surfactants.